STUDIES ON EHRLICHIA CHAFFEENSIS AND BORRELIA LONESTARI, TICK-BORNE AGENTS TRANSMITTED BY AMBLYOMMA AMERICANUM by ANDREA SOARES VARELA (Under the Direction of Susan E. Little) ABSTRACT Over the past decade, the lone star tick (LST), Amblyomma americanum, has become recognized as an important vector of several known or suspected zoonotic agents in the southern United States. Among these are Ehrlichia chaffeensis, the causative agent of human monocytotropic ehrlichiosis, E. ewingii, and agent of human and canine ehrlichiosis, and Borrelia lonestari, the putative agent of “southern tick- associated rash illness” (STARI). White-tailed deer (WTD) are a primary host for all motile LST stages and serve as the principal reservoir host for E. chaffeensis. However, studies that reveal the course of E. chaffeensis infection in reservoir hosts are scarce. Reports of E. ewingii and B. lonestari in LST from the southern U.S. exist but additional reports would add to an understanding of the distribution of these bacteria. In addition, B. lonestari has not yet been isolated in culture, making the development of specific diagnostic assays to differentiate STARI from Lyme disease problematical. This dissertation presents studies which investigated these three tick-borne agents. Findings in Chapter 3 demonstrated that the course of primary infection with E. chaffeensis via different routes of needle inoculation did not differ. Chapter 4 showed that WTD with a primary infection of E. chaffeensis were not protected from secondary infection with a genetic variant. In Chapter 5, inoculation of domestic goats with E. chaffeensis did not cause active infection, suggesting that goats are an unsuitable model for E. chaffeensis infection. In Chapter 6, a three-year survey of LST from northeastern Georgia found prevalences of E chaffeensis, E. ewingii, and B. lonestari at 2.0% (8/398), 4.8% (19/398), and 1.0% (4/398), respectively, substantiating the presence of these organisms and risk of human exposure in northeastern Georgia. The detection of B. lonestari in LST from northeastern Georgia was critical for obtaining the first culture isolate of B. lonestari. The isolation and molecular, immunologic, and ultramicroscopic characterization used to identify the spirochetes are described in Chapter 6. These studies contribute to an understanding of recently recognized bacteria transmitted by LST. INDEX WORDS: Amblyomma americanum, Borrelia lonestari, Ehrlichia chaffeensis, Ehrlichia ewingii, experimental infection, lone star tick, Odocoileus virginianus, white-tailed deer STUDIES ON EHRLICHIA CHAFFEENSIS AND BORRELIA LONESTARI, TICK-BORNE AGENTS TRANSMITTED BY AMBLYOMMA AMERICANUM by ANDREA SOARES VARELA BS, Cook College, Rutgers University, 1997 DVM, Tufts University, 2001 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2004 © 2004 Andrea Soares Varela All Rights Reserved STUDIES ON EHRLICHIA CHAFFEENSIS AND BORRELIA LONESTARI, TICK-BORNE AGENTS TRANSMITTED BY AMBLYOMMA AMERICANUM by ANDREA SOARES VARELA Major Professor: Susan E. Little Committee: Elizabeth W. Howerth Ray M. Kaplan David E. Stallknecht William R. Davidson Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August 2004 iv DEDICATION This work is dedicated to my mother and father, Ana and Eduardo, who nurtured and encouraged my desires to pursue an extensive academic career. v ACKNOWLEDGEMENTS This dissertation would not have been possible without gracious assistance and support from many people, including my committee members, technicians, graduate students, friends, and family. My mentor, Dr. Susan Little deserves the greatest acknowledgment for her patience, encouragement, and foremost, her ability to open doors to opportunities that I may not have otherwise experienced. Dr. Randy Davidson was an amazing resource in both wildlife disease and parasitology, and his frequent witticisms were refreshing to my usual cynicism. Dr. Dave Stallknecht was always available for the “quick” culture question, the final word in experiment decisions, and “big-picture” questions. This work could not have been done without the pathology expertise and resources of Dr. Buffy Howerth who took the time to teach me techniques and look at results, despite her incredibly busy schedule. Finally, Dr. Ray Kaplan was an essential link to the practical side of veterinary parasitology. All of my committee members were fundamental in helping me to develop into a veterinary parasitologist. I am also greatly thankful to the graduate and vet students, research scientists, and technicians, both in the department and at SCWDS, who provided help and motivation in this research and in maintaining some sanity. Among these are friends and colleagues, Michael Yabsley, Gus Moore, Molly Murphy, and Cynthia Tate. In addition, Dana Ambrose and Page Luttrell became close dear friends and provided enormous support, both emotionally and directly towards this research. Finally, I am deeply grateful to my family, and especially John Stokes, now my lifelong companion. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ............................................................................................................v CHAPTER 1 INTRODUCTION........................................................................................................ 1 2 LITERATURE REVIEW............................................................................................. 9 3 EXPERIMENTAL INFECTION OF WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS) WITH EHRLICHIA CHAFFEENSIS BY DIFFERENT INOCULATION ROUTES...................................................................................62 4 PRIMARY AND SECONDARY INFECTION WITH EHRLICHIA CHAFFEENSIS IN WHITE-TAILED DEER.....................................................76 5 ATTEMPTED INFECTION OF GOATS WITH EHRLICHIA CHAFFEENSIS.99 6 DISEASE AGENTS IN AMBLYOMMA AMERICANUM FROM NORTHEASTERN GEORGIA ........................................................................ 110 7 FIRST CULTURE ISOLATION OF BORRELIA LONESTARI, PUTATIVE AGENT OF SOUTHERN TICK-ASSOCIATED RASH ILLNESS...............136 8 CONCLUSIONS .....................................................................................................166 REFERENCES.........................................................................................................................172 1 CHAPTER 1 INTRODUCTION Amblyomma americanum, the lone star tick (LST), is a three-host ixodid tick prevalent throughout the southeastern and south-central United States. Within the past twenty years, the role of LST has escalated from that of a pest to one of public health significance. While important as a cause of irritation and blood loss during high infestations, LST have also become appreciated in recent years as efficient vectors of emerging human pathogens. In the 1990s alone, three newly-recognized human pathogens, Ehrlichia chaffeensis, E. ewingii, and Borrelia lonestari, which cause human monocytotropic ehrlichiosis (HME), ehrlichiosis in canines and rarely humans, and southern tick-associated rash illness (STARI) in humans, respectively, have been implicated as transmitted by LST (Anziani, et al. 1990, Ewing, et al. 1995, Barbour, et al. 1996, Means and White 1997, Buller, et al. 1999). Of these three agents, the natural history of E. chaffeensis is most completely known. Ehrlichia chaffeensis has been isolated from humans and white-tailed deer (WTD), the principal natural reservoir host, on multiple occasions (Dawson, et al. 1991, Lockhart, et al. 1997a, Lockhart, et al. 1997b, Little, et al. 1998, Paddock, et al. 2001, Yabsley, et al. 2003b). Studies of E. chaffeensis infection dynamics in WTD, however, are limited. Primary infection in deer has been reported four times via either intravenous injection or tick transmission (Dawson, et al. 1994b, Ewing, et al. 1995, 2 Kocan, et al. 2000a, Davidson, et al. 2001). In addition, the importance of molecular variation of different genetic types in influencing the virulence and epizootiology of E. chaffeensis is not understood. A closely related organism, E. ewingii is considered primarily a pathogen of dogs. This organism has been implicated in less than ten human ehrlichiosis cases, primarily in immunocompromised patients (Buller, et al. 1999, Paddock, et al. 2001). Ehrlichia ewingii has not yet been isolated in culture, but has been detected by PCR or microscopy in humans, ticks, and various mammals including WTD in the South (Anderson, et al. 1992, Dawson, et al. 1996a, Goldman, et al. 1998, Murphy, et al. 1998, Buller, et al. 1999, Paddock, et al. 2001, Arens, et al. 2003, Childs and Paddock 2003). Until 2003, B. lonestari had been identified through molecular detection in ticks, deer, and a human patient (Burkot, et al. 2001, James, et al. 2001, Bacon, et al. 2003, Moore, et al. 2003a, Stegall-Faulk, et al. 2003, Stromdahl, et al. 2003, Yabsley, et al. 2003). Suspected cases of STARI have been widely reported in the South, where classic Lyme disease is rare (Campbell, et al. 1995, Masters and Donnell 1995, Kirkland, et al. 1997, Masters, et al. 1998, Armstrong, et al. 2001). The studies presented contribute to a growing understanding of the disease agents transmitted by A. americanum, particularly E. chaffeensis and B. lonestari. Specific aims of the research for this dissertation were as follows: 1. Monitor progression of primary infection